The invention described herein was made in part in the course of work under a grant or award from the United States Army, No. DAMD 17-86-C-6038.
As a mechanism of self defense, animals have developed a complex set of responses to foreign material, collectively called the immune system. Immune responses are generally advantageous (protective) in nature, however, under certain situations, the animal body produces an immune response that is undesirable. Examples of such undesirable responses include allergic reactions, characterized by the production of IgE antibodies to extrinsic antigens, and autoimmune diseases in which the immune system reacts against self antigens.
During the past few decades, a number of methods have been described for inhibiting, suppressing or xe2x80x9ccuringxe2x80x9d specific immune responses. These methods involve the treatment of animals with different kinds of chemical preparations, the details of which are described below. The immune modification methodology which forms the basis of the present series of applications is based on the premise that the immune system recognizes foreign antigens in the context of physically constrained arrays. In order to stimulate the immune system, arrays must exceed a specific size (or geometry) and have a minimum number of physically accessible epitopes which are identical in nature (minimum valence). Once these two parameters are met or exceeded, the immune system will respond by the production of antibodies (IgM, IgG and/or IgE) by antigen specific B-cells and by the production of T-cell factors and/or activities (T-cell xe2x80x98helpxe2x80x99, cytokines, cytoxicity, etc.).
The method to which the present invention relates is based on the finding by Applicants that this system can be manipulated by introducing synthetically derived macromolecular arrays that are xe2x80x9csubthresholdxe2x80x9d in geometry and/or valence and that are designed to compete with naturally occurring arrays for the suppression of autoimmune and extrinsic allergic responses.
The technology which forms the basis of the invention is derived from the Immunon model of immune response described by Dintzis et al in Proc. Nat""l. Acad. Sci. USA, 73:3671-3675 (1976). That paper discloses the concept of there being a threshold as to the number and spacing of haptens on T-cell independent antigens in order to obtain an immunogenic response. The 1976 paper also discloses that the non-immunogenic polymers are suppressive of the action of immunogenic polymers towards triggering the de novo immune response in non-immunized animals. The suppressive effect of non-immunogenic polymers on the immunogenic response of immunogenic polymers is further described in Proc. Nat""l. Acad. Sci. USA, 79:395, 1982; Proc. Nat""l. Acad. Sci. USA, 79:884, 1982; and J. Immunol., 131:2196, 1983. (See also Dintzis et al, J. Immunol. 135:423, 1985; Dintzis et al In: Theoretical Immunology, Pt. 1, Vol. II. ed. Perelson, A. S. Addison-Wesley Publishing Co., Reading, Mass. pp 83-103, 1988; Dintzis et al, J. Immunol. 143:1239, 1989; Dintzis et al, Eur. J. Immunol. 70:229, 1990; and Dintzis and Dintzis, Immunol. Reviews No. 115, pp 243-251, 1990).
The earlier applications of the present series include details of studies that were done using experimental paradigms involving T-independent antibody responses which can be assessed by the level of IgM production. The use of size restricted backbones of various types (linear polyacrylamide, dextran, Ficoll, carboxymethyl cellulose, etc.) to suppress IgM antibody production to small molecular weight haptens such as DNP and fluorescein is specifically described. (See Examples 1 to 7 below.) In addition, reference is made in the earlier filings to the use of the present invention to suppress allergies to pollen and auto-immune disease, including multiple sclerosis and myasthemia gravis. The present application includes details of studies relating to T-cell dependent antibody production as well as T-cell responses by themselves. The data presented herein thus further support the applicability of the immune suppression methodology of the earlier filed applications in this series to complex responses involving T-cell dependent antibody production, represented by IgG and IgE. In addition, the present disclosure underscores the desirability of characterizing the suppressive constructs to ensure that they are free from potentially simulatory molecules.
As indicated above, varying chemical preparations reportedly suitable for use in methods of inhibiting immune responses have been the subject of numerous publications. The methods disclosed are apparently based on the xe2x80x9cspecial chemical compositionxe2x80x9d of the polymeric backbone material used which forms an epitope carrier. The mechanisms by which the observed specific immune suppression occurs, and the specific molecular attributes inferred to bring about the suppression, have been variously ascribed to:
1) chemical composition as determined by the ratios of carbon to hydrogen to oxygen in the carrier material (Dawn et al, J. Immunol. 126:407-413, (1981); Wei et al, Int. Archs. Allergy Appl. Immunol. 85:1-7 (1988)). 2) xe2x80x9cunnaturalnessxe2x80x9d as defined by the use of the xe2x80x9cunnaturalxe2x80x9d D-amino acids, rather than xe2x80x9cnaturalxe2x80x9d L-amino acids in synthesizing the polypeptide carrier substance (Katz et al, J. Exp. Med., 134:201-223 (1971); Liu et al, Proc. Natl. Acad. Sci. USA 76:1430-1434 (1979); Liu et al, J. Allergy Clin. Immunol. 66:322-326 (1980));
3) xe2x80x9cspecialxe2x80x9d chemical properties, undefined in nature; and
4) ability to increase xe2x80x9cspecific suppressor cellsxe2x80x9d in undefined ways. (See specific comments that follow). To the best of Applicants"" knowledge, however, no other group has proposed that immune suppression occurs because the suppressive material contains molecules with the proper combination of molecular size and epitope valence and, thus, no other group has taught or even suggested the method to which the present invention relates.
Sehon and coworkers have carried out a number of studies on specific immune suppression, induced byte injection of polymeric molecules composed of epitopes coupled to a polyvinylalcohol (PVA) backbone structure (see, for example, Dawn et al, J. Immunol. 126:407-413 (1981); Wei et al, Int. Archs. Allergy Appl. Immunol. 85:1-7 (1988)). The PVA backbone structure was created by reacting low molecular weight PVA, 14 kDa, with cyanogen bromide to convert some of the hydroxyl groups on, the polymer to a reactive form, and coupling those activated hydroxyl groups to amino groups on aliphatic diamine. This reaction was expected by the authors to substitute the PVA polymer molecules with a number of free aliphatic amino groups from the unreacted ends of the diamine adduct. These ends were subsequently substituted with hapten groups to form multiply substituted PVA molecules of molecular weight supposedly almost unchanged from that of the original PVA.
This empirical procedure produced soluble haptenated polymeric material which was suppressive of specific immune responses against the hapten involved. However, in reacting a multiply reactive polymer (cyanogen bromide activated PVA) with an excess of a divalent reactant (ethylenediamine) a very substantial amount of cross-linkage between the polymer molecules occurred with the resulting formation of multiply cross-linked molecules of a wide range of molecular weights. Although Sehon and Lee noted that precipitates formed, and discarded them, they apparently did not take this as an indication that higher molecular weight (and thus potentially stimulatory) polymers were being produced.
Applicants have, in fact, reported, (Dintzis et al, J. Immunol. 143:1239-1244 (1989)) that higher molecular weight (over 100 kDa) PVA molecules multiply substituted with hapten are immunogenic in vivo and in vitro, giving bell shaped dose response curves. Similar molecules with molecular weights below 100 kDa, however, were found by Applicants to be inhibitory of the immune response, without having stimulatory capacity, as predicted by their paradigm.
Katz and co-workers have described the specific suppression of the immune response to epitopes by treatment with polymer preparations composed of those epitopes coupled to a carrier backbone made of the synthetic polypeptide, poly(D-glutamic acid, D-lysine) or poly(D-Glu,D-Lys) (see, for example, (Katz et al, J. Exp. Med. 134:201-223 (1971); Liu et al, Proc. Natl. Acad. Sci. USA 76:1430-1434 (1979); Liu et al J. Allergy Clin. Immunol. 66:322-326 (1980)). This polypeptide is a commercially available randomly ordered polymer synthesized from chemically activated forms of the D-amino acids, D-lysine and D-glutamic acid, in the ratio 60:40. Katz has rationalized the findings of immune suppression as caused by the xe2x80x9cunnaturalxe2x80x9d character of the synthetic polypeptide composed of the unusual D-amino acids rather than the usual L- forms of the amino acids, which are found in all protein molecules. This interpretation was apparently supported by the finding that equivalent immune suppression was not observed when the carrier backbone polypeptide was synthesized from the more normal L-amino acids.
The findings of Katz fit well into the Immunon paradigm as illustrated below:
1) The poly(D-Glu,D-Lys) preparation used by Katz as a backbone polymer was obtained from commercial sources, and had average molecular weight of less than 100 kDa (the primary commercial producers, Yeda (in Israel) and Sigma (in St. Louis), have informed Applicants that it is not possible for them to produce such polymers with average molecular weights greater than 70 kDa). Thus, Katz apparently used polymers of molecular weight less than 100 kDa as suppressive backbone material, without realizing the significance of this fortuitous choice of molecular weight.
2) Starting with the highest molecular weight poly(D-Glu,D-Lys) available, approximately 70 kDa from Yeda, Applicants substituted a number of the lysine amino groups with the hapten, fluorescein and found the resulting FLU-poly(D-Glu,D-Lys) to be non-immunogenic, as expected. Examination of the material by HPLC revealed that, as expected, it contained molecules with a wide range of molecular weights, from under 40 kDa to a small amount over 100 kDa. When size fractionated by gel filtration chromatography on Superose CL-6B columns, it was possible to separate out a small amount of material of molecular weight approximately 200 kDa. This higher molecular weight fraction proved to be immunogenic for an immune response against fluorescein in mice. This finding indicates that there is nothing intrinsically suppressive about FLU-poly(D-Glu,D-Lys), but that it can be stimulatory or non-stimulatory, depending on the molecular size.
3) To further test the effect of molecular size, Applicants cross-linked non-immunogenic 70 kDa FLU-poly(D-Glu,D-Lys) molecules with carbodiimide, coupling some carboxyl groups on glutamic acid residues with amino groups on lysine residues to form stable amide bonds. A wide range of molecular weight products resulted. When these were size-fractionated on gel filtration columns, the material with molecular weights well above 100 kDa were immunogenic both in vivo and in vitro, whereas the fractions with molecular weights under 100 kDa was not immunogenic. This again fits the expectations of the Immunon hypothesis, and is not consistent with the interpretations put forth by Katz.
4) Since the mammalian body does not produce enzymes capable of hydrolyzing polypeptides composed solely of D-amino acids, it is to be expected that such polypeptides, whether free or epitope substituted, will not be rapidly degraded in the animal body, and will be long lasting in their effects. However, polypeptides made of the usual L-amino acids can be rapidly hydrolyzed by normal proteolytic enzymes and would not be expected to have sustained effects. This suggests that the properties ascribed by Katz to the xe2x80x9cunnaturalxe2x80x9d nature of the D-amino acid polypeptide are due solely to the resistance to enzymatic breakdown, a characteristic shared by many synthetic and natural polymeric molecules,
Diener and co-workers have published a number of papers describing the specific suppressive immune effects of epitopes coupled to carboxy-methylcellulose as carrier (see, for example, Diner et al, J. Immunol. 122:1886-1891 (1979)). These have been ascribed by Diener to the special chemical nature of carboxymethyl cellulose, without consideration of the molecular weight of the material. However, Applicants have reported that haptenated preparations of carboxymethyl cellulose of molecular weights under 100 kDa are suppressive for epitopes coupled to them, without being stimulatory at any dose, whereas preparations of molecular weights over 100 kDa are stimulatory at proper doses (Dintzis et al, J. Immunol. 143:1239-1244 (1989)). Apparently, Diener used material of molecular weight predominately under 100 kDa, without realizing the significance that the molecular size of the polymers had on the immune effects of his preparations.
The specific suppressive effect of hapten coupled to polyvinylpyrrolidone (PVP), a material which has been used as a blood substitute has been reported (von Specht et al, Clin. Exp. Immunol. 33:292-297 (1978); Lee et al, Eur. J. Immunol. 11:13-17 (1981)). Other authors have published on similar suppressive effects of haptens coupled to Ficoll (Watanabe et al, J. Immunol. 118:251-255 (1977)), pneumococcal polysaccharides (Borel et al, Nature 261:49-50 (1976); Mitchell et al, Eur. J. Immunol. 2:460-467 (1972)), plant polysaccharides (Moreno et al, Clin. Exp. Immunol. 31:499-511 (1978)); Humphrey, Eur. J. Immunol. 11:212-220 (1981)) or isologous immunoglobulin (Lee et al, J. Immunol. 114:829-842 (1975); Borel et al, Nature 261:49-50 (1976)). These reports are quite diverse, but do not address the combined effects of the molecular weight of polymer carrier and the epitope valence on the immune response which results from their administration, as Applicants have done. Molecular weight characterization of the epitope-substituted polymer preparations was not done in these published studies. However, the experimental protocols are consistent with the interpretation that the average molecular weights of these preparations was under 100 kDa in all instances.
In general, authors who have reported specific suppressive effects from hapten-coupled polymer preparations have apparently chanced upon preparations which fit Applicants"" description of suppressive soluble molecules, namely a substantial number of epitopes coupled to a soluble polymeric carrier of molecular weight less than about 100 kDa. While these conditions may be unwittingly encountered under a variety of circumstances, as noted above, such encounters are not suggestive of the present invention.